Methods and compositions for detection of cancer

ABSTRACT

A molecular probe for use in detection of cancer cells expressing an Ig superfamily cell adhesion molecule that binds in a homophilic fashion in a subject includes a targeting agent that specifically binds to and/or complexes with a proteolytically cleaved extracellular fragment of the Ig superfamily cell adhesion molecule.

RELATED APPLICATION

This application claims priority from U.S. Provisional Application Nos.60/088,955, filed Aug. 14, 2008 and 61/170,850 filed Apr. 20, 2009, thesubject matter of which are incorporated herein by reference in theirentirety.

GOVERNMENT FUNDING

This invention was made with government support under Grant No. NS051520awarded by The National Institutes of Health. The United Statesgovernment has certain rights in the invention.

BACKGROUND

The prognosis for high-grade brain tumors, such as glioblastomamultiforme (GBM) is extremely poor with a median survival of about oneyear from diagnosis (Ichimura K, Ohgaki H, Kleihues P, Collins V P(2004) Molecular pathogenesis of astrocytic tumours. J Neurooncol70:137-160; Louis D N, Ohgaki H, Wiestler O D, Cavenee W K (2007) WorldHealth Organization Classification of Tumours of the Nervous System, 4thEdition. Lyon: IARC). Several biological characteristics contribute tothe lethality of GBM tumors, including their uncontrolled proliferationin the restricted cranial space, and their highly dispersive nature(Ichimura K, Ohgaki H, Kleihues P, Collins V P (2004) Molecularpathogenesis of astrocytic tumours. J Neurooncol 70:137-160; Louis D N,Ohgaki H, Wiestler O D, Cavenee W K (2007) World Health OrganizationClassification of Tumours of the Nervous System, 4th Edition. Lyon:IARC; Louis D N (2006) Molecular pathology of malignant gliomas. AnnuRev Pathol 1:97-117). Surgical resection remains the primary treatmentfor glial tumors (Furnari F B, Fenton T, Bachoo R M, Mukasa A, Stommel JM, Stegh A, Hahn W C, Ligon K L, Louis D N, Brennan C, Chin L, DePinho RA, Cavenee W K (2007) Malignant astrocytic glioma: genetics, biology,and paths to treatment. Genes Dev 21:2683-2710) and more completeresection has been linked to improved survival (Sanai N, Berger M S(2008) Glioma extent of resection and its impact on patient outcome.Neurosurgery 62:753-764; discussion 264-756). However, by the time ofdiagnosis, GBM cells have usually dispersed extensively into thesurrounding brain, making it difficult for the surgeon to preciselylocalize the tumor margin (Nakada M, Nakada S, Demuth T, Tran N L,Hoelzinger D B, Berens M E (2007) Molecular targets of glioma invasion.Cell Mol Life Sci 64:458-478). Magnetic resonance imaging (MRI) guidedstereotactic techniques are typically utilized to maximize resection.However, MRI is limited in its ability to detect sparse tumor cellsinvading surrounding normal brain (Sorensen A G, Batchelor T T, Wen P Y,Zhang W T, Jain R K (2008) Response criteria for glioma. Nat Clin PractOncol 5:634-644). Since nearly all glioblastomas recur locally, betterdetection would likely improve surgical resection resulting in enhancedpatient survival.

SUMMARY

The present invention relates to a molecular probe for use in detectionof cancer cells expressing an immunoglobulin (Ig) superfamily celladhesion molecule that includes an extracellular homophilic bindingportion, which can bind in homophilic fashion or engage in homophilicbinding in a subject. The molecular probe includes a targeting agentthat specifically binds to and/or complexes with a proteolyticallycleaved extracellular fragment of the Ig superfamily cell adhesionmolecule.

In one aspect of the invention, the Ig superfamily cell adhesionmolecule can include a cell surface receptor protein tyrosinephosphatase (PTP) type IIb, such PTPμ or a PTPμ like molecule. Thecancer cell can be a glioma cell and, specifically, a glioblastomamultiforme (GBM) cell.

In another aspect of the invention, the extracellular fragment can havean amino acid sequence of SEQ ID NO: 2. The targeting agent canspecifically bind to and/or complex with SEQ ID NO: 2. In a furtheraspect, the targeting agent can bind to homophilic binding domains orportion of the extracellular fragment, such as SEQ ID NO: 3, whichcomprises the MAM, Ig and first two FNIII repeat binding domain of PTPμ.

In yet another aspect of the invention, the targeting agent can includea peptide having an amino acid sequence that is substantially homologousto about 10 to about 50 consecutive amino acids of the amino acidsequence of SEQ ID NO: 3. Examples of peptides having an amino acidsequence substantially homologous to SEQ ID NO: 3 can be peptides havingan amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7.

In another aspect of the invention, the molecular probe can include adetectable moiety that is linked to the targeting agent. The molecularprobe can be detected in vivo by recognizing the detectable moiety. Thedetectable moiety can be detected by at least one of gamma imaging,positron emission tomography (PET) imaging, computer tomography (CT)imaging, magnetic resonance imaging, near infrared imaging, orfluorescent imaging.

The present invention also relates to a method of detecting cancer cellsexpressing an Ig superfamily cell adhesion molecule that binds in ahomophilic fashion in a subject. The method includes administering amolecular probe to the subject. The molecular probe can include atargeting agent that specifically binds to and/or complexes with aproteolytically cleaved extracellular fragment of the Ig superfamilycell adhesion molecule and imaging agent linked to the targeting agent.The molecular probe bound to and/or complexed with the proteolyticallycleaved extracellular fragment of the Ig superfamily cell adhesionmolecule is detected in the subject to provide the location and/ordistribution of the cancer cells in the subject.

In an aspect of the invention, the Ig superfamily cell adhesion moleculecan include a cell surface receptor protein tyrosine phosphatase (PTP)type IIb, such PTPμ or a PTPμ like molecule. The cancer cell can be aglioma cell and, specifically, a glioblastoma multiforme (GBM) cell.

In another aspect of the invention, the extracellular fragment can havean amino acid sequence of SEQ ID NO: 2. The targeting agent canspecifically bind to and/or complex with SEQ ID NO: 2. In a furtheraspect, the targeting agent can bind to homophilic binding domains orportion of the extracellular fragment, such as SEQ ID NO: 3, whichcomprises the MAM and Ig binding domain of PTPμ.

In yet another aspect of the invention, the targeting agent can includea peptide having an amino acid sequence that is substantially homologousto about 10 to about 50 consecutive amino acids of the amino acidsequence of SEQ ID NO: 3. Examples of peptides having an amino acidsequence substantially homologous to SEQ ID NO: 3 can be peptides havingan amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7.

The molecular probe can detected in vivo by detecting the detectablemoiety. The detectable moiety can be detected by at least one of gammaimaging, positron emission tomography (PET) imaging, computer tomography(CT) imaging, magnetic resonance imaging, near infrared imaging, orfluorescent imaging.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a series of immunoblots showing a 55 kDaextracellular fragment of PTPμ is detected in human glioblastoma tissue.(A) GBM tumors from three patients were divided into center (ctr) andedge samples, lysed, separated by SDS-PAGE and immunoblotted using anantibody against the MAM domain of PTPμ. Noncancerous normal cortex(ctx) from the same patients was loaded for comparison. Equal proteinload was verified by stripping and reprobing the immunoblot with anantibody to vinculin. (B) LN-229 or Gli36Δ5 xenograft flank tumorprotein extracts were immunoblotted as above. Human GBM tumor tissue (T)was loaded on the same blot for comparison.

FIG. 2 is a schematic illustration of a PTPμ structure and targetingpeptide probe sequences. PTPμ is a transmembrane protein that mediatesefficient cell-cell adhesion via the MAM domain, Ig domain and FNIIIrepeats within its extracellular segments. (A) The scissors indicate theapproximate site where PTPμ is cleaved to generate a 55 kDa N-terminalfragment. The sequence is shown for the PTPμ MAM and Ig domains (i.e.,SEQ ID NO: 2). The highlighted regions indicate the sequences used togenerate PTPμ peptide probes (i.e., SBK1 (SEQ ID NO: 4), SBK2 (SEQ IDNO: 5), SBK3 (SEQ ID NO: 6), and SBK4 (SEQ ID NO: 7). (B) Crystalstructure of the Ig and MAM domains of PTPμ (PDB ID:2V5Y). SBK1 (SEQ IDNO: 4) and SBK2 (SEQ ID NO: 5) were derived from the N-terminal MAMdomain while SBK3 (SEQ ID NO: 6) and SBK4 (SEQ ID NO: 7) were from theIg domain.

FIG. 3 illustrates images showing SBK2 peptide (SEQ ID NO: 5) probespecifically recognizes human glioblastoma tissue but not normal brain.Sections of noncancerous normal cortical brain tissue from epilepsypatients or GBM tumor were histochemically labeled with TexasRed-conjugated SBK1 (SEQ ID NO: 4), SBK2 (SEQ ID NO: 5) or ScrambledSBK2 peptide. Two examples of normal and GBM tissue are shown that arerepresentative of 6 different samples examined.

FIG. 4 illustrates images showing SBK3 (SEQ ID NO: 6) and SBK4 (SEQ IDNO: 7) peptide probes specifically recognize human glioblastoma tissuebut not normal brain. Sections of noncancerous normal cortical braintissue from epilepsy patients or GBM tumor were histochemically labeledwith Texas Red-conjugated SBK3 (SEQ ID NO: 6), SBK4 (SEQ ID NO: 7) orScrambled SBK2 peptide. Two examples of normal and GBM tissue are shownthat are representative of 6 different samples examined.

FIG. 5 illustrates images showing PTPμ mAb blocks SBK2 (SEQ ID NO: 5)and SBK4 (SEQ ID NO: 7) peptide binding to human glioblastoma tissue.Preincubation of a PTPμ extracellular antibody raised against the MAMdomain (BK2) specifically blocked SBK2-TR (SEQ ID NO: 5) and SBK4-TR(SEQ ID NO: 7) peptide binding to GBM tumor tissue sections.

FIG. 6 illustrates images showing small cell clusters from LN-229 flanktumors label with PTPμ peptide SBK2 (SEQ ID NO: 5). Flank tumors ofLN-229 cells were excised, fixed and sectioned. The LN-229 cells expressGFP. Binding of the Texas Red-conjugated SBK2 peptide is shown in twotumor samples. SBK2 peptide (SEQ ID NO: 5) labels small clusters ofcells in the tumor microenvironment.

FIG. 7 illustrates images showing PTPμ peptides SBK2 (SEQ ID NO: 5) andSBK4 (SEQ ID NO: 7) recognize Gli36Δ5 mouse flank tumors in vivo. TexasRed (TR) or Alexa (AL)-conjugated PTPμ peptides were administeredintravenously to mice with xenograft flank tumors of Gli36Δ5 cells. (A)Fluorescent images of SBK2-AL peptide labeling. Panel labeled “beforetx” (before time course) shows the animal autofluorescent background.The tumor cells are expressing GFP. (B) Time course of peptide bindingto flank tumors (N=3 animals tested per peptide). Average normalizedsignals acquired in the tumor region of interest were plotted. The errorbars represent standard error of the mean from the 3 animals. (C)Regions of interest shown over the tumor and non-tumor skin. (D) Brightfield image of the flank tumor labeled in (A).

FIG. 8 illustrates images showing PTPμ peptides SBK2 (SEQ ID NO: 5) andSBK4 (SEQ ID NO: 7) recognize LN-229 mouse flank tumors in vivo. TexasRed (TR) or Alexa (AL)-conjugated PTPμ peptides were administeredintravenously to mice with xenograft flank tumors of LN-229 cells. (A)Fluorescent images of SBK2-AL peptide labeling. Panel labeled “beforetx” (before time course) shows the animal autofluorescent background.The tumor cells are expressing GFP. (B) Time course of peptide bindingto flank tumors (N=3 animals tested per peptide). Average normalizedsignals acquired in the tumor region of interest were plotted. The errorbars represent standard error of the mean from the 3 animals. (C)Regions of interest shown over the tumor and non-tumor skin. (D) Brightfield image of the flank tumor labeled in (A).

FIG. 9 illustrates PTPμ peptide SBK2 (SEQ ID NO: 5) labels Gli36Δ5intracranial tumors in vivo. Texas Red (TR) or Alexa (AL)-conjugatedPTPμ peptides were administered intravenously to mice with xenograftintracranial tumors of Gli36Δ5 cells. (A) Bright field images of brainslices containing Gli36Δ5-GFP tumor. (C) GFP fluorescence of the slicesshown in (A), indicating the location of the Gli36Δ5-GFP tumor in eachslice. (B) Alexa-750 fluorescence images of the slices shown in (A),indicating binding of the PTPμ peptide SBK2 (SEQ ID NO: 5). Furthermore,these data suggest that SBK2 crosses the blood brain barrier to detectthe glioma cells. (D) Fluorescence overlay image showing both GFP andAlexa-750 fluorescence signals. (E) Bright field image of brain slicesfrom a control brain following tail vein injection of SBK2-AL peptide.(F) Alexa-750 fluorescence image of the slices shown in (E), indicatingthat SBK2-AL peptide does not bind non-tumor brain. (G) Fluorescencequantitation of peptide binding in brain slices containing Gli36Δ5-GFPtumor cells labeled with SBK2-AL peptide (n=6), scrambled SBK2-ALpeptide (n=7), or PBS (n=6). Quantitation of SBK2-AL labeling of controlbrain slices (n=4) is also shown.

FIG. 10 illustrates images showing Gli36Δ5 flank tumors label with PTPμpeptides. Flank tumors of Gli36Δ5 cells were excised, fixed andsectioned. The Gli36Δ5 cells express GFP. Binding of the TexasRed-conjugated SBK2 (SEQ ID NO: 5) and SBK4 peptides (SEQ ID NO: 7) isshown. The peptides label both the tumor cells and the extracellularspaces in the tumor microenvironment.

FIG. 11 illustrates images showing LN-229 flank tumors label with PTPμpeptides. Flank tumors of LN-229 cells were excised, fixed andsectioned. The LN-229 cells express GFP. Binding of the TexasRed-conjugated SBK2 (SEQ ID NO: 5) and SBK4 peptides (SEQ ID NO: 7) isshown. The peptides label both the tumor cells and the extracellularspaces in the tumor microenvironment.

DETAILED DESCRIPTION

Methods involving conventional molecular biology techniques aredescribed herein. Such techniques are generally known in the art and aredescribed in detail in methodology treatises, such as Current Protocolsin Molecular Biology, ed. Ausubel et al., Greene Publishing andWiley-Interscience, New York, 1992 (with periodic updates). Unlessotherwise defined, all technical terms used herein have the same meaningas commonly understood by one of ordinary skill in the art to which thepresent invention pertains. Commonly understood definitions of molecularbiology terms can be found in, for example, Rieger et al., Glossary ofGenetics: Classical and Molecular, 5th Edition, Springer-Verlag: NewYork, 1991, and Lewin, Genes V, Oxford University Press: New York, 1994.

“Antibody” or “antibody peptide(s)” refer to an intact antibody, or abinding fragment thereof that competes with the intact antibody forspecific binding. Binding fragments are produced by recombinant DNAtechniques, or by enzymatic or chemical cleavage of intact antibodies.Binding fragments include Fab, Fab′, F(ab′)₂, Fv, and single-chainantibodies. An antibody other than a “bispecific” or “bifunctional”antibody is understood to have each of its binding sites identical. Anantibody substantially inhibits adhesion of a polypeptide to a specificbinding partner when an excess of antibody reduces the quantity of thepolypeptide bound to the specific binding partner by at least about 20%,40%, 60% or 80%, and more usually greater than about 85% (as measured inan in vitro competitive binding assay).

The term “monoclonal” refers to an antibody that specifically binds to asequence of amino acid and/or a specific epitope of an antigen.

The term “polyclonal” refers to an antibody that recognizes multipleepitope sites on a single antigen.

The term “epitope” includes any protein determinant capable of specificbinding to an immunoglobulin. Epitope determinants usually consist ofchemically active surface groupings of molecules such as amino acids orsugar side chains and usually have specific three-dimensional structuralcharacteristics, as well as specific charge characteristics.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, or an extract madefrom biological materials.

The terms “patient”, “subject”, “mammalian host,” and the like are usedinterchangeably herein, and refer to mammals, including human andveterinary subjects.

The terms “peptide(s)”, “protein(s)” and “polypeptide(s)” are usedinterchangeably herein. As used herein, “polypeptide” refers to anypeptide or protein comprising two or more amino acids joined to eachother by peptide bonds or modified peptide bonds (i.e., peptideisomers). “Polypeptide(s)” refers to both short chains, commonlyreferred as peptides, oligopeptides or oligomers, and to longer chainsgenerally referred to as proteins.

The terms “polynucleotide sequence” and “nucleotide sequence” are alsoused interchangeably herein.

“Recombinant,” as used herein, means that a protein is derived from aprokaryotic or eukaryotic expression system

The term “wild type” refers to the naturally-occurring polynucleotidesequence encoding a protein, or a portion thereof, or protein sequence,or portion thereof, respectively, as it normally exists in vivo.

The term “mutant” refers to any change in the genetic material of anorganism, in particular a change (i.e., deletion, substitution,addition, or alteration) in a wild type polynucleotide sequence or anychange in a wild type protein. The term “variant” is usedinterchangeably with “mutant”. Although it is often assumed that achange in the genetic material results in a change of the function ofthe protein, the terms “mutant” and “variant” refer to a change in thesequence of a wild type protein regardless of whether that change altersthe function of the protein (e.g., increases, decreases, imparts a newfunction), or whether that change has no effect on the function of theprotein (e.g., the mutation or variation is silent).

As used herein, the term “nucleic acid” refers to polynucleotides, suchas deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA). The term should also be understood to include, as equivalents,analogs of either RNA or DNA made from nucleotide analogs, and, asapplicable to the embodiment being described, single (sense orantisense) and double-stranded polynucleotides.

As used herein, the term “gene” or “recombinant gene” refers to anucleic acid comprising an open reading frame encoding a polypeptide,including both exon and (optionally) intron sequences.

“Homology” and “identity” are used synonymously throughout and refer tosequence similarity between two peptides or between two nucleic acidmolecules. Homology can be determined by comparing a position in eachsequence, which may be aligned for purposes of comparison. When aposition in the compared sequence is occupied by the same base or aminoacid, then the molecules are homologous or identical at that position. Adegree of homology or identity between sequences is a function of thenumber of matching or homologous positions shared by the sequences.

A “chimeric protein” or “fusion protein” is a fusion of a first aminoacid sequence encoding a polypeptide with a second amino acid sequencedefining a domain (e.g. polypeptide portion) foreign to and notsubstantially homologous with any domain of the first polypeptide. Achimeric protein may present a foreign domain which is found (albeit ina different protein) in an organism which also expresses the firstprotein, or it may be an “interspecies”, “intergenic”, etc. fusion ofprotein structures expressed by different kinds of organisms.

The term “isolated” as used herein with respect to nucleic acids, suchas DNA or RNA, refers to molecules separated from other DNAs, or RNAs,respectively, which are present in the natural source of themacromolecule. The term isolated as used herein also refers to a nucleicacid or peptide that is substantially free of cellular material, orculture medium when produced by recombinant DNA techniques, or chemicalprecursors or other chemicals when chemically synthesized. Moreover, an“isolated nucleic acid” is meant to include nucleic acid fragments,which are not naturally occurring as fragments and would not be found inthe natural state.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intraventricular, intracapsular, intraorbital, intracardiac,intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal and intrasternalinjection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, agent or other materialother than directly into a specific tissue, organ, or region of thesubject being treated (e.g., brain), such that it enters the animal'ssystem and, thus, is subject to metabolism and other like processes, forexample, subcutaneous administration.

The present invention relates to a molecular probe for use in detectionof cancer cells expressing an Ig superfamily cell adhesion molecule thatincludes an extracellular homophilic binding portion or segment, whichbinds in a homophilic fashion or engages in homophilic binding in asubject. The molecular probe of the present invention can beadministered systemically to a subject and readily cross the blood brainbarrier to define cancer metastases or tumor cell margin in the subject.

It was found that metastic cancer cell migration or cancer celldispersal, such as glioblastoma multiforme (GBM) cell dispersal, canoccur along characteristic pathways of anatomical structures (e.g.,brain), which are rich in cell adhesion molecules (CAMs) andextracellular matrix molecules (ECM) that are permissive substrates forcell migration. In some instances, cancer cell dispersal, such as GBMcell dispersal, requires the production of proteolytic enzymes, whichgives the cell the ability to move through its environment. For example,GBM cells overexpress growth factor receptor protein tyrosine kinasesand their ligands, which is an important prerequisite for tumor growthand dispersal. The activity of the receptor tyrosine kinases is normallykept in check by the opposing activity of protein tyrosine phosphatases,such as receptor protein tyrosine phosphatases (RPTPs) (e.g., PTPμ),which are thought to be important regulators of adhesion-dependentsignals. Receptor protein tyrosine phosphatases, such as RPTP type IIbcell adhesion molecules, have been shown to regulate neural developmentand axon guidance. Ensslen-Craig S E, Brady-Kalnay S M (2004) Receptorprotein tyrosine phosphatases regulate neural development and axonguidance. Dev Biol 275:12-22; Tonks N K (2006) Protein tyrosinephosphatases: from genes, to function, to disease. Nat Rev Mol Cell Biol7:833-846).

RPTP type IIb cell adhesion molecules can include an extracellularsegment that engages in homophilic binding. For example, theextracellular fragment of PTPμ, which is expressed by GBM cells, caninclude a MAM domain, an immunoglobulin (Ig) domain and four fibronectintype III (FNIII) repeats. PTPμ binds homophilically (i.e., the “ligand”for PTPμ is an identical PTPμ molecule on an adjacent cell) and canmediate cell-cell aggregation. The Ig domain of PTPμ is responsible forpromoting homophilic interactions and proper cell surface localization.The MAM domain also plays an important role in cell adhesion andsorting. The first two FNIII repeats contribute to efficient celladhesion. When expressed on the cell surface, PTPμ mediates cell-celladhesion and transduces signals in response to adhesion that mayregulate contact inhibition of movement.

In at least some human cancer cells that express Ig superfamily celladhesion molecules that include an extracellular segment, which engagesin homophilic binding, (e.g., PTPμ) the extracellular fragment isproteolytically cleaved and found to associate with or localize to thecancer cell margin or surface. It was found that molecular probes, thatcan specifically bind to and/or complex with these proteolyticallycleaved extracellular fragments or segments can be used to detect cancancer cell migration, tumor cell dispersal, tumor cell invasion anddefine cancer metastases and tumor margins in a subject.

By way of example, it was determined that proteolytically cleaved PTPμextracellular fragments are common to high-grade glioblastomas.Molecular probes to the PTPμ fragment were shown to clearly demarcatethe tumor cells in tissue sections and the PTPμ extracellular fragmentis present in human tumor “edge” samples, suggesting that the molecularprobe can be used as diagnostic tools for molecular imaging ofdispersive brain tumors or the tumor margin. Systemic introduction ofmolecular probes in accordance with the present invention resulted inrapid and specific labeling of the flank tumors within minutes. Labelingoccurred primarily within the tumor, however a gradient of molecularprobe at the tumor margin was also observed.

One aspect of the present invention therefore relates to a method ofdetecting cancer cells expressing an Ig superfamily cell adhesionmolecule that binds in a homophilic fashion in a subject by detecting aproteolytically cleaved extracellular fragment of the Ig superfamilycell adhesion molecule with a molecular probe. In one example, the Igsuperfamily cell adhesion molecule that engages in homophilic bindingcan include RPTP type IIb cell adhesion molecules. In another example,Ig superfamily cell adhesion molecules that engage in homophilic bindingcan include RPTPs of the PTPμ-like subfamily, such as PTPμ, PTPκ, PTPρ,and PCP-2 (also called PTPλ).

PTPμ-like RPTPs include a MAM (Meprin/A5-protein/PTPμ) domain, an Igdomain, and FNIII repeats. PTPμ can have the amino acid sequence of SEQID NO: 1, which is identified by Genbank Accession No. AAI51843.1. Itwill be appreciated that the PTPμ gene can generate splice variants suchthat the amino acid sequence of PTPμ can differ from SEQ ID NO: 1. Insome embodiments of the invention, PTPμ can have an amino acid sequenceidentified by Genbank Accession No. AAH51651.1 and Genbank Accession No.AAH40543.1.

Cancer cells that express an Ig superfamily cell adhesion molecule thatbinds in a homophilic fashion and that can be proteolytically cleaved toproduce a detectable extracellular fragment can include, for example,metastic or motile cancer cells. In one example, the invasive,dispersive, motile or metastic cancer cells can include glioma cells.The term glioma, as used herein, refers to a type of cancer arising fromglial cells in the brain or spine. Gliomas as contemplated by thepresent invention can be classified by cell type, by tumor grade, and/orby location. For example, ependymomas resemble ependymal cells,astrocytomas (e.g. glioblastoma multiforme) resemble astrocytes,oligodedrogliomas resemble oligodendrocytes. Also mixed gliomas, such asoligoastrocytomas may contain cells from different types of glia.Gliomas can also be classified according to whether they are above orbelow a membrane in the brain called the tentorium. The tentoriumseparates the cerebrum, above, from the cerebellum, below. Asupratentorial glioma is located above the tentorium, in the cerebrum,and occurs mostly in adults whereas an infratentorial glioma is locatedbelow the tentorium, in the cerebellum, and occurs mostly in children.Other examples of cancer cells that express an Ig superfamily celladhesion molecule that binds in a homophilic fashion can be readilydetermined by using, for example, immunoassays.

The molecular probe that is used to detect the extracellular fragment ofthe proteolytically cleaved extracellular fragment of the Ig superfamilycell adhesion molecule that engages in homophilic binding can include atargeting agent that specifically binds to and/or complexes with theproteolytically cleaved extracellular fragment of the Ig superfamilycell adhesion molecule of the cancer cell. The targeting agent caninclude a targeting small molecule, peptide, or antibody that binds toand/or complexes with the proteolytically cleaved extracellular fragmentof the Ig superfamily cell adhesion molecule and that can readily beadministered to the subject using, for example, parenteral or systemicadministration techniques (e.g., intravenous infusion).

In one aspect of the invention, the targeting agent can include apeptide or targeting peptide that binds to and/or complexes with theproteolytically cleaved extracellular fragment of the Ig superfamilycell adhesion molecule. The targeting peptide can have an amino acidsequence that is substantially homologous to about 10 to about 50consecutive amino acids of a homophilic binding portion or domain of theproteleolytically cleaved extracellular fragment of the Ig superfamilycell adhesion molecule. By substantially homologous, it is meant thetargeting peptide has at least about 80%, about 90%, about 95%, about96%, about 97%, about 98%, about 99% or about 100% sequence identitywith a portion of the amino acid sequence of the homophilic bindingportion of the proteleolytically cleaved extracellular fragment of theIg superfamily cell adhesion molecule.

In one example, the homophilic binding portion of the Ig superfamilycell adhesion molecule can include, for example, the Ig domain of thecell adhesion molecule. In another example, where the Ig superfamilycell adhesion molecule is PTPμ, the homophilic binding portion caninclude the Ig binding domain and the MAM domain.

In another aspect of the invention, the targeting peptide can have anamino acid sequence that is substantially homologous to about 10 toabout 50 consecutive amino acids of the Ig binding domain and/or MAMdomain of PTPμ (e.g., SEQ ID NO: 1) and readily cross the blood brainbarrier when systemically administered to a subject. The development ofthe PTPμ targeting peptides can be based on a large body of structuraland functional data. The sites required for PTPμ-mediated homophilicadhesion have been well characterized. In addition, the crystalstructure of PTPμ can provide information regarding which regions ofeach functional domain are likely to be exposed to the outsideenvironment and therefore available for homophilic binding and thusdetection by a peptide probe.

In yet another aspect of the invention, the proteolytically cleavedextracellular fragment of PTPμ (e.g., SEQ ID NO: 1) can include an aminoacid sequence of SEQ ID NO: 2, the Ig and MAM binding region cancomprise the amino acid sequence of SEQ ID NO: 3, and targeting peptidecan have an amino acid sequence that is substantially homologous toabout 10 to about 50 consecutive amino acids of SEQ ID NO: 2 or SEQ IDNO: 3. Examples of targeting peptide that can specifically bind SEQ IDNO: 2 or SEQ ID NO: 3 can have an amino acid sequence selected from thegroup consisting of SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO: 6, and SEQ IDNO: 7. Targeting peptides comprising SEQ ID NO: 4 or 5 can recognize orbind to the MAM domain; whereas targeting peptides comprising SEQ ID NO:6 or 7 can recognize or bind to the Ig domain. Targeting peptidescomprising SEQ ID NO: 4, 5, 6 or 7 can recognize or bind to the MAM, Igdomain or the FNIII repeats.

The targeting peptides in accordance with the present invention can besubject to various changes, substitutions, insertions, and deletionswhere such changes provide for certain advantages in its use. In thisregard, targeting peptides that binds to and/or complex with aproteolytically cleaved extracellular portion of an Ig superfamily celladhesion molecule can correspond to or be substantially homologous with,rather than be identical to, the sequence of a recited peptide where oneor more changes are made and it retains the ability to function asspecifically binding to and/or complexing with the proteolyticallycleaved extracellular portion of an Ig superfamily cell adhesionmolecule.

The targeting peptide can be in any of a variety of forms of peptidederivatives, that include amides, conjugates with proteins, cyclizedpeptides, polymerized peptides, analogs, fragments, chemically modifiedpeptides, and the like derivatives.

The term “analog” includes any peptide having an amino acid residuesequence substantially identical to a sequence specifically shown hereinin which one or more residues have been conservatively substituted witha functionally similar residue and that specifically binds to and/orcomplexes with the proteolytically cleaved extracellular portion of anIg superfamily cell adhesion molecule as described herein. Examples ofconservative substitutions include the substitution of one non-polar(hydrophobic) residue, such as isoleucine, valine, leucine or methioninefor another, the substitution of one polar (hydrophilic) residue foranother, such as between arginine and lysine, between glutamine andasparagine, between glycine and serine, the substitution of one basicresidue such as lysine, arginine or histidine for another, or thesubstitution of one acidic residue, such as aspartic acid or glutamicacid for another.

The phrase “conservative substitution” also includes the use of achemically derivatized residue in place of a non-derivatized residueprovided that such peptide displays the requisite binding activity.

“Chemical derivative” refers to a subject peptide having one or moreresidues chemically derivatized by reaction of a functional side group.Such derivatized molecules include for example, those molecules in whichfree amino groups have been derivatized to form amine hydrochlorides,p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonylgroups, chloroacetyl groups or formyl groups. Free carboxyl groups maybe derivatized to form salts, methyl and ethyl esters or other types ofesters or hydrazides. Free hydroxyl groups may be derivatized to formO-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine maybe derivatized to form N-im-benzylhistidine. Also included as chemicalderivatives are those peptides, which contain one or more naturallyoccurring amino acid derivatives of the twenty standard amino acids. Forexamples: 4-hydroxyproline may be substituted for proline;5-hydroxylysine may be substituted for lysine; 3-methylhistidine may besubstituted for histidine; homoserine may be substituted for serine; andornithine may be substituted for lysine. Peptides of the presentinvention also include any peptide having one or more additions and/ordeletions or residues relative to the sequence of a peptide whosesequence is shown herein, so long as the requisite activity ismaintained.

The term “fragment” refers to any subject peptide having an amino acidresidue sequence shorter than that of a peptide whose amino acid residuesequence is shown herein.

Additional residues may also be added at either terminus of a peptidefor the purpose of providing a “linker” by which the peptides of thisinvention can be conveniently affixed to a detectable moiety, label,solid matrix, or carrier.

Amino acid residue linkers are usually at least one residue and can be40 or more residues, more often 1 to 10 residues. Typical amino acidresidues used for linking are tyrosine, cysteine, lysine, glutamic andaspartic acid, or the like. In addition, a subject polypeptide candiffer by the sequence being modified by terminal-NH2 acylation, e.g.,acetylation, or thioglycolic acid amidation, byterminal-carboxylamidation, e.g., with ammonia, methylamine, and thelike terminal modifications. Terminal modifications are useful, as iswell known, to reduce susceptibility by proteinase digestion, andtherefore serve to prolong half life of the polypeptides in solutions,particularly biological fluids where proteases may be present. In thisregard, polypeptide cyclization is also a useful terminal modification,and is particularly preferred also because of the stable structuresformed by cyclization and in view of the biological activities observedfor such cyclic peptides as described herein.

Any peptide or compound of the present invention may also be used in theform of a pharmaceutically acceptable salt. Acids, which are capable offorming salts with the peptides of the present invention, includeinorganic acids such as trifluoroacetic acid (TFA) hydrochloric acid(HCl), hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid,sulfuric acid, phosphoric acetic acid, propionic acid, glycolic acid,lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid,maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalenesulfonic acid, sulfanilic acid or the like.

Bases capable of forming salts with the peptides of the presentinvention include inorganic bases such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide and the like; and organic bases such asmono-, di- and tri-alkyl and aryl-amines (e.g., triethylamine,diisopropylamine, methylamine, dimethylamine and the like) andoptionally substituted ethanolamines (e.g. ethanolamine, diethanolamineand the like).

Targeting peptides of the present invention can be synthesized by any ofthe techniques that are known to those skilled in the polypeptide art,including recombinant DNA techniques. Synthetic chemistry techniques,such as a solid-phase Merrifield-type synthesis, can be used for reasonsof purity, antigenic specificity, freedom from undesired side products,ease of production and the like. A summary of the many techniquesavailable can be found in Steward et al., “Solid Phase PeptideSynthesis”, W. H. Freeman Co., San Francisco, 1969; Bodanszky, et al.,“Peptide Synthesis”, John Wiley & Sons, Second Edition, 1976; J.Meienhofer, “Hormonal Proteins and Peptides”, Vol. 2, p. 46, AcademicPress (N.Y.), 1983; Merrifield, Adv. Enzymol., 32:221-96, 1969; Fieldset al., int. J. Peptide Protein Res., 35:161-214, 1990; and U.S. Pat.No. 4,244,946 for solid phase peptide synthesis, and Schroder et al.,“The Peptides”, Vol. 1, Academic Press (N.Y.), 1965 for classicalsolution synthesis, each of which is incorporated herein by reference.Appropriate protective groups usable in such synthesis are described inthe above texts and in J. F. W. McOmie, “Protective Groups in OrganicChemistry”, Plenum Press, New York, 1973, which is incorporated hereinby reference.

In general, the solid-phase synthesis methods contemplated comprise thesequential addition of one or more amino acid residues or suitablyprotected amino acid residues to a growing peptide chain. Normally,either the amino or carboxyl group of the first amino acid residue isprotected by a suitable, selectively removable protecting group. Adifferent, selectively removable protecting group is utilized for aminoacids containing a reactive side group such as lysine.

Using a solid phase synthesis as an example, the protected orderivatized amino acid can be attached to an inert solid support throughits unprotected carboxyl or amino group. The protecting group of theamino or carboxyl group can then be selectively removed and the nextamino acid in the sequence having the complimentary (amino or carboxyl)group suitably protected is admixed and reacted under conditionssuitable for forming the amide linkage with the residue already attachedto the solid support. The protecting group of the amino or carboxylgroup can then be removed from this newly added amino acid residue, andthe next amino acid (suitably protected) is then added, and so forth.After all the desired amino acids have been linked in the propersequence, any remaining terminal and side group protecting groups (andsolid support) can be removed sequentially or concurrently, to affordthe final linear polypeptide.

It will be appreciated that the targeting peptide can bind to and/orcomplex with homophilic binding domains of proteolytically cleavedextracellular fragments of other Ig superfamily cell adhesion molecules,besides PTPs. For example, a similar molecular detection strategydescribed herein can be used with any other Ig superfamily cell adhesionmolecule having a homophilic binding cell surface protein whose ligandbinding site is known. A large variety of cell surface proteins,including other phosphatases, are cleaved at the cell surface (StreuliM, Krueger N, Ariniello P, Tang M, Munro J, Blattler W, Adler D,Disteche C, Saito H (1992) Expression of the receptor-linked proteintyrosine phosphatase LAR: proteolytic cleavage and shedding of theCAM-like extracellular region. EMBO J. 11:897-907; Anders L, Mertins P,Lammich S, Murgia M, Hartmann D, Saftig P, Haass C, Ullrich A (2006)Furin-, ADAM 10-, and gamma-secretase-mediated cleavage of a receptortyrosine phosphatase and regulation of beta-catenin's transcriptionalactivity. Mol Cell Biol 26:3917-3934; Haapasalo A, Kim D Y, Carey B W,Turunen M K, Pettingell W H, Kovacs D M (2007)Presenilin/gamma-secretase-mediated cleavage regulates association ofleukocyte-common antigen-related (LAR) receptor tyrosine phosphatasewith beta-catenin. J Biol Chem 282:9063-9072; Chow J P, Fujikawa A,Shimizu H, Noda M (2008) Plasmin-mediated processing of protein tyrosinephosphatase receptor type Z in the mouse brain. Neurosci Lett442:208-212). These proteins represent additional targets for that canbe readily used by the skilled artisan for forming molecular probes thatcan be used to detect cancers (Barr A J, Ugochukwu E, Lee W H, King O N,Filippakopoulos P, Alfano I, Savitsky P, Burgess-Brown N A, Muller S,Knapp S (2009) Large-scale structural analysis of the classical humanprotein tyrosine phosphatome. Cell 136:352-363). Furthermore, thetargeting peptides can be used as a starting point to develop higheraffinity small molecules, antibodies, and/or antibody fragments withsimilar ligand binding capabilities. The development and screening ofsmall molecules from pharmacophores of the targeting peptides using, forexample, in silico screening, can be readily performed, and the bindingaffinity of such identified molecules can be readily screened againsttargeting peptides using assays described herein to select smallmolecule targeting agents.

In certain aspects of the invention, the targeting agent is directly orindirectly labeled with a detectable moiety. The role of a detectablemoiety is to facilitate the detection step of a diagnostic method byallowing visualization of the complex formed by binding of the molecularprobe to the proteolytically cleaved extracellular fragment of the Igsuperfamily cell adhesion molecule. The detectable moiety can beselected such that it generates a signal, which can be measured andwhose intensity is related (preferably proportional) to the amount ofthe molecular probe bound to the tissue being analyzed. Methods forlabeling biological molecules, such as polypeptides and antibodies arewell-known in the art.

Any of a wide variety of detectable moieties can be used in the practiceof the present invention. Examples of detectable moieties include, butare not limited to: various ligands, radionuclides, fluorescent dyes,chemiluminescent agents, microparticles (such as, for example, quantumdots, nanocrystals, phosphors and the like), enzymes (such as, forexample, those used in an ELISA, i.e., horseradish peroxidase,beta-galactosidase, luciferase, alkaline phosphatase), colorimetriclabels, magnetic labels, and biotin, dioxigenin or other haptens andproteins for which antisera or monoclonal antibodies are available.

In some aspects of the invention, the molecular probes described hereinmay be used in conjunction with non-invasive imaging (e.g.,neuroimaging) techniques for in vivo imaging of the molecular probe,such as magnetic resonance spectroscopy (MRS) or imaging (MRI), or gammaimaging, such as positron emission tomography (PET) or single-photonemission computed tomography (SPECT). The term “in vivo imaging” refersto any method, which permits the detection of a labeled molecular probe,as described above. For gamma imaging, the radiation emitted from theorgan or area being examined is measured and expressed either as totalbinding or as a ratio in which total binding in one tissue is normalizedto (for example, divided by) the total binding in another tissue of thesame subject during the same in vivo imaging procedure. Total binding invivo is defined as the entire signal detected in a tissue by an in vivoimaging technique without the need for correction by a second injectionof an identical quantity of molecular probe along with a large excess ofunlabeled, but otherwise chemically identical compound.

For purposes of in vivo imaging, the type of detection instrumentavailable is a major factor in selecting a given detectable moiety. Forinstance, the type of instrument used will guide the selection of thestable isotope. The half-life should be long enough so that it is stilldetectable at the time of maximum uptake by the target, but short enoughso that the host does not sustain deleterious effects.

In one example, the detectable moiety can include a radiolabel that isdetected using gamma imaging wherein emitted gamma irradiation of theappropriate wavelength is detected. Methods of gamma imaging include,but are not limited to, SPECT and PET. For SPECT detection, the chosenradiolabel can lack a particular emission, but will produce a largenumber of photons in, for example, a 140-200 keV range. For PETdetection, the radiolabel can be a positron-emitting moiety, such as19F.

In another example, the detectable moiety can an include MRS/MRIradiolabel, such as gadolinium, 19F, 13C, that is coupled (e.g.,attached or complexed) with the targeting agent using general organicchemistry techniques. The detectable moiety can also includeradiolabels, such as 18F, 11C, 75Br, or 76Br for PET by techniques wellknown in the art and are described by Fowler, J. and Wolf, A. inPOSITRON EMISSION TOMOGRAPHY AND AUTORADIOGRAPHY (Phelps, M., Mazziota,J., and Schelbert, H. eds.) 391-450 (Raven Press, NY 1986) the contentsof which are hereby incorporated by reference. The detectable moiety canalso include 1231 for SPECT. The 1231 can be coupled to the targetingagent can by any of several techniques known to the art. See, e.g.,Kulkarni, Int. J. Rad. Appl. & Inst. (Part B) 18: 647 (1991), thecontents of which are hereby incorporated by reference. In addition,detectable moiety can include any radioactive iodine isotope, such as,but not limited to 1311, 1251, or 1231. The radioactive iodine isotopescan be coupled to the targeting agent by iodination of a diazotizedamino derivative directly via a diazonium iodide, see Greenbaum, F. Am.J. Pharm. 108: 17 (1936), or by conversion of the unstable diazotizedamine to the stable triazene, or by conversion of a non-radioactivehalogenated precursor to a stable tri-alkyl tin derivative which thencan be converted to the iodo compound by several methods well known tothe art.

The detectable moiety can further include known metal radiolabels, suchas Technetium-99m (99 mTc). Modification of the targeting agent tointroduce ligands that bind such metal ions can be effected withoutundue experimentation by one of ordinary skill in the radiolabeling art.The metal radiolabeled molecular probes can then be used to detectcancers, such as GBM in the subject. Preparing radiolabeled derivativesof Tc99m is well known in the art. See, for example, Zhuang et al.,“Neutral and stereospecific Tc-99m complexes: [99mTc]N-benzyl-3,4-di-(N-2-mercaptoethyl)-amino-pyrrolidines (P-BAT)”Nuclear Medicine & Biology 26(2):217-24, (1999); Oya et al., “Small andneutral Tc(v)O BAT, bisaminoethanethiol (N2S2) complexes for developingnew brain imaging agents” Nuclear Medicine & Biology 25(2):135-40,(1998); and Hom et al., “Technetium-99m-labeled receptor-specificsmall-molecule radiopharmaceuticals: recent developments and encouragingresults” Nuclear Medicine & Biology 24(6):485-98, (1997).

The molecular probe can be administered to the subject by, for example,systemic, topical, and/or parenteral methods of administration. Thesemethods include, e.g., injection, infusion, deposition, implantation, ortopical administration, or any other method of administration whereaccess to the tissue by the molecular probe is desired. In one example,administration of the molecular probe can be by intravenous injection ofthe molecular probe in the subject. Single or multiple administrationsof the probe can be given. “Administered”, as used herein, meansprovision or delivery of a molecular probe in an amount(s) and for aperiod of time(s) effective to label cancer cells in the subject.

Molecular probes of the present invention can be administered to asubject in a detectable quantity of a pharmaceutical compositioncontaining a molecular probe or a pharmaceutically acceptablewater-soluble salt thereof, to a patient. A “detectable quantity” meansthat the amount of the detectable compound that is administered issufficient to enable detection of binding of the compound to the cancercells. An “imaging effective quantity” means that the amount of thedetectable compound that is administered is sufficient to enable imagingof binding of the molecular probe to the cancer cells.

The molecular probes administered to a subject can be used to determinethe presence, location, and/or distribution of cancer cells, i.e.,cancer cells associated with proteolytically cleaved extracellularfragments of Ig superfamily cell adhesion molecules, in an organ or bodyarea, such as the brain, of a patient. The presence, location, and/ordistribution of the molecular probe in the animal's tissue, e.g., braintissue, can be visualized (e.g., with an in vivo imaging modalitydescribed above). “Distribution” as used herein is the spatial propertyof being scattered about over an area or volume. In this case, “thedistribution of cancer cells” is the spatial property of cancer cellsbeing scattered about over an area or volume included in the animal'stissue, e.g., brain tissue. The distribution of the molecular probe maythen be correlated with the presence or absence of cancer cells in thetissue. A distribution may be dispositive for the presence or absence ofa cancer cells or may be combined with other factors and symptoms by oneskilled in the art to positively detect the presence or absence ofmigrating or dispersing cancer cells, cancer metastases or define atumor margin in the subject.

In one aspect of the invention, the molecular probes of the presentinvention may be administered to a subject to assess the distributionGBM cells in a subject's brain and correlate the distribution to aspecific location. Neurosurgeons routinely use stereotactic techniquesand intra-operative MRI (iMRI) in surgical resections. This allows themto specifically identify and sample tissue from distinct regions of thetumor such as the tumor edge or tumor center. Frequently, they alsosample regions of brain on the tumor margin that are outside the tumoredge that appear to be grossly normal but are infiltrated by dispersingtumor cells upon histological examination.

Molecular probes in accordance with the present invention thatspecifically bind to and/or complex with proteolytically cleaved Igsuperfamily cell adhesion molecules (PTPμ) associated with GBM cells canbe used in intra-operative imaging techniques to guide neurosurgicalresection and eliminate the “educated guess” of the location of thetumor margin by the neurosurgeon. Previous studies have determined thatmore extensive surgical resection improves patient survival Stummer W,Novotny A, Stepp H, Goetz C, Bise K, Reulen H J (2000)Fluorescence-guided resection of glioblastoma multiforme by using5-aminolevulinic acid-induced porphyrins: a prospective study in 52consecutive patients. J Neurosurg 93:1003-1013. Fluorescence-guidedresection of glioblastoma multiforme by using 5-aminolevulinicacid-induced porphyrins: a prospective study in 52 consecutive patients.Stummer W, Novotny A, Stepp H, Goetz C, Bise K, Reulen H J (2000)Fluorescence-guided resection of glioblastoma multiforme by using5-aminolevulinic acid-induced porphyrins: a prospective study in 52consecutive patients. J Neurosurg 93:1003-1013. Thus, molecular probesthat function as diagnostic molecular imaging agents have the potentialto increase patient survival rates.

In accordance with another aspect of the invention, the methods andmolecular probes described herein can be used to monitor and/or comparethe migration, dispersal, and metastases of a cancer in a subject priorto administration of a cancer therapeutic, during administration of acancer therapeutic, or post therapeutic regimen.

A “cancer therapeutic” or “cancer therapy”, as used herein, can includeany agent or treatment regimen that is capable of negatively affectingcancer in an animal, for example, by killing cancer cells, inducingapoptosis in cancer cells, reducing the growth rate of cancer cells,reducing the incidence or number of metastases, reducing tumor size,inhibiting tumor growth, reducing the blood supply to a tumor or cancercells, promoting an immune response against cancer cells or a tumor,preventing or inhibiting the progression of cancer, or increasing thelifespan of an animal with cancer. Cancer therapeutics can include oneor more therapies such as, but not limited to, chemotherapies, radiationtherapies, hormonal therapies, and/or biologicaltherapies/immunotherapies. A reduction, for example, in cancer volume,growth, migration, and/or dispersal in a subject may be indicative ofthe efficacy of a given therapy. This can provide a direct clinicalefficacy endpoint measure of a cancer therapeutic. Therefore, in anotheraspect of the present invention, a method of monitoring the efficacy ofa cancer therapeutic is provided. More specifically the presentinvention provides for a method of monitoring the efficacy of a cancertherapy.

The method of monitoring the efficacy of a cancer therapeutic caninclude the steps of administering in vivo to the animal a molecularprobe as described herein, then visualizing a distribution of themolecular probe in the animal (e.g., with an in vivo imaging modality asdescribed herein), and then correlating the distribution of themolecular probe with the efficacy of the cancer therapeutic. It iscontemplated that the administering step can occur before, during, andafter the course of a therapeutic regimen in order to determine theefficacy of a chosen therapeutic regimen. One way to assess the efficacyof the cancer therapeutic is to compare the distribution of a molecularprobe pre and post cancer therapy.

In certain embodiments of the invention, the methods and molecularprobes of the present invention can be used to measure the efficacy oftherapeutic administered to a subject for treating glioblastomamultiforme. In this embodiment, the molecular probe can be administeredto the subject prior to, during, or post administration of thetherapeutic regimen and the distribution of glioblastoma multiformecells can be imaged to determine the efficacy of the therapeuticregimen. In one example, the therapeutic regimen can include a surgicalresection of the glioblastoma multiforme and the molecular probe can beused to define the distribution of the glioblstoma multiformepre-operative and post-operative to determine the efficacy of thesurgical resection. Optionally, the methods and molecular probes of thepresent invention can be used in an intra-operative surgical procedure,such as a surgical tumor resection, to more readily define and/or imagethe cancer cell mass or volume during the surgery.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples, which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example

We recently found that normal brain and primary rat astrocytes expressPTPμ, but the most dispersive glial tumors, GBMs, downregulate PTPμexpression. The downregulation of PTPμ occurs via proteolytic cleavagein human GBM tumors, which may be an important event that dysregulatesnormal contact inhibition of movement. In this example, it isdemonstrated that PTPμ proteolytic cleavage generates an extracellularfragment of PTPμ in human GBM tumors that is retained in the tumormicroenvironment. This extracellular fragment contains the domainsrequired for efficient PTPμ-mediated homophilic cell-cell adhesion. Wedevised a strategy to detect this PTPμ extracellular fragment bygenerating a series of fluorescently tagged peptide probes tosurface-exposed sites of PTPμ, based on crystallographic data (AricescuA R, Hon W C, Siebold C, Lu W, van der Merwe P A, Jones E Y (2006)Molecular analysis of receptor protein tyrosine phosphatase mu-mediatedcell adhesion. Embo J 25:701-712.; Aricescu A R, Siebold C, Choudhuri K,Chang V T, Lu W, Davis S J, van der Merwe P A, Jones E Y (2007)Structure of a tyrosine phosphatase adhesive interaction reveals aspacer-clamp mechanism. Science 317:1217-1220.; Aricescu A R, Siebold C,Jones E Y (2008) Receptor protein tyrosine phosphatase micro: measuringwhere to stick. Biochem Soc Trans 36:167-172). The peptide probesspecifically recognized primary human GBM cells in tissue sections ofsurgically resected tumor. More importantly, the peptides recognized GBMtumors in vivo in two different human GBM xenografts in nude mice, andare capable of crossing the blood-brain bather to label intracranial GBMtumors. These results indicate that the cleaved PTPμ extracellularfragment remains associated with the GBM microenvironment and is aunique biomarker of GBM cells that can be used as a molecular diagnosticimaging agent to detect the tumor margin of human glioblastomas in vivo.

Materials and Methods

Peptide Synthesis and Conjugation

SBK1 (SEQ ID NO: 4), SBK2 (SEQ ID NO: 5), SBK3 (SEQ ID NO: 6) and SBK4(SEQ ID NO: 7) peptides were synthesized either on an Applied Biosystemsmodel 433A synthesizer or were purchased from Genscript Corporation(Piscataway, N.J., USA). Following synthesis, the N-terminal glycineresidue of each peptide was specifically coupled to Texas Red-X (mixedisomers) or Alexa-750 succinimidyl ester dye (Molecular Probes Inc.,Eugene, Oreg.), which has a five-carbon spacer between the succinimidegroup that couples to the N-terminal amine and the fluorophore.

Human Brain Tissue Protein Extraction

Using a FDA-approved computer navigational device and software (BrainLabVector Vision 2 and i-Plan 2.0) the neurosurgeon co-registered multiplescalp points with volumetric RAGE T1±Gadolinium MRI (1.5 mm segments/0skips) obtained the day prior to surgery using a standard technique(Z-Touch). After achieving precision of <1 mm and confirmation usingvarious objective skull landmarks, surgery was performed usingstereotactic techniques. After pathological confirmation of GBM wasobtained, the stereotactic device was used to identify the edge andcenter of the Gadolinium enhanced tumor mass and paired specimens ofapproximately 100 mg each were preserved in liquid nitrogen orformalin-paraffin, respectively. In some cases, non-eloquent brain wasalso identified and similarly preserved if it was part of the region tobe resected. Similarly, discarded tissue from patients undergoingcortical resections for intractable epilepsy was collected fornoncancerous “normal” tissue after the neuropathologists released thesample. Following tissue resection, the samples were snap frozen, thawedon ice in lysis buffer and protein was extracted as previouslydescribed. Mouse flank Gli36Δ5 or LN-229 tumors were excised, snapfrozen, then lysed using a PRO 200 tissue tearor (PRO Scientific Inc.,Monroe, Conn., USA). All samples were separated by SDS-PAGE andtransferred to nitrocellulose for immunoblotting with antibodies againstthe extracellular segment of PTPμ (BK9) (Brady-Kalnay S M, Tonks N K(1994) Identification of the homophilic binding site of the receptorprotein tyrosine phosphatase PTPμ. J Biol Chem 269:28472-28477).

Peptide Labeling of Human Brain Tissue

Human glioblastoma or noncancerous “normal” epilepsy tissue samples wereimmediately fixed in 10% neutral buffered formalin (Sigma-Aldrich, USA).Tissues were ethanol dehydrated and paraffin embedded. Tissue sectionswere cut at 5 μm intervals and stored at room temperature (RT). Beforestaining, tissue sections were deparaffinized and blocked with 2% goatserum in PBS for 20 minutes at RT. PTPμ-TR peptides were diluted inblock buffer and incubated with the tissue sections for 1 hour at RT inthe dark. The sections were rinsed with PBS, coverslipped with CitifluorAntifadent Mounting Medium, AF1 (Electron Microscopy Sciences, Hatfield,Pa.) and imaged immediately at 40× on a Nikon TE-200 invertedmicroscope, using a SPOT-RT camera and SPOT software version 3.2(Diagnostic Instruments, Inc., Sterling Heights, Mich.). Highmagnification phase and fluorescent images were taken using the sameexposure settings between multiple experiments. The workingconcentrations for the peptides were determined empirically for tissuestaining and are as follows: SBK1-TR, 40 μM; SBK2-TR, 10 μM; SBK3-TR, 10μM; and SBK4-TR, 3.3 μM. The SBK4-TR peptide was the most effective inlabeling tissue sections.

Antibody Blocking Experiments

To block PTPμ binding sites, human tumor tissue sections werepre-incubated for 1 hour at RT with BK2 monoclonal antibody, raisedagainst the MAM domain of PTPμ (Brady-Kalnay S M, Tonks N K (1994)Identification of the homophilic binding site of the receptor proteintyrosine phosphatase PTPμ. J Biol Chem 269:28472-28477), prior toincubation with SBK2-TR (10 μM) or SBK4-TR (3.3 μM) peptide in blockbuffer for 1 hour at RT in the dark. Tissue sections were rinsed andimaged as described above.

Heterotopic Xenograft Tumors

Human Gli36Δ5 glioblastoma cells constitutively over-express the vIIImutant forms of the EGFR gene (Tyminski E, Leroy S, Terada K,Finkelstein D M, Hyatt J L, Danks M K, Potter P M, Saeki Y, Chiocca E A(2005) Brain tumor oncolysis with replication-conditional herpes simplexvirus type 1 expressing the prodrug-activating genes, CYP2B 1 andsecreted human intestinal carboxylesterase, in combination withcyclophosphamide and irinotecan. Cancer Res 65:6850-6857). Human LN-229glioblastoma cells were obtained from American Type Culture Collection,Manassas, Va. Gli36Δ5 or LN-229 cells were harvested for flankimplantation by trypsinization. In some experiments, the cells wereinfected with lentivirus to express GFP (Tyagi M, Karn J (2007) CBF-1promotes transcriptional silencing during the establishment of HIV-1latency. EMBO J. 26:4985-4995) 48 hours prior to harvesting. The cells(2×106 cells for flank tumor implants) were re-suspended in a 1:1dilution of PBS and Matrigel (BD Biosciences; Franklin Lakes, N.J.) fora total volume of 250-300 μL per flank tumor implant per animal.

NIH athymic nude female mice (5-8 weeks and 20-25 g upon arrival,NCl—NIH) were obtained. All animal protocols were IACUC approved. Forflank tumor implants, mice were anesthetized with inhaledisofluorane:oxygen for immobilization. The Matrigel: cell mixture wasloaded into a 1 mL syringe fitted with a 26-gauge needle and kept onice. The mixture was injected subcutaneously in the right flank regionof the mouse.

Peptide Labeling of Mouse Flank Tumor Sections

NIH athymic nude female mice implanted with Gli36Δ5 or LN-229 flanktumors for 2 to 3 weeks were anesthetized with inhaled isoflurane:oxygen and sacrificed by decapitation. Flank tumors were then excised,fixed in 4% paraformaldehyde in PEM buffer (80 mM Pipes, 5 mM EGTA, 1 mMMagnesium Chloride, 3% sucrose), pH 7.4, embedded in OCT andcryosectioned at 5 μm intervals. Slides were stored at −20° C. Forpeptide labeling experiments, the slides were thawed and incubated withpeptide diluted to 100-200 μM as described above.

In Vivo Imaging of Flank Tumors

Nude mice with Gli36Δ5 or LN-229 flank tumors were imaged at 9-28 daysafter cell injection. Fluorophore-conjugated PTPμ peptides were dilutedto 100 μM (SBK2-Alexa) or 200 μM (SBK1-TR, SBK3-TR, SBK4-TR, ScrambledSBK2-TR control peptide) in PBS and injected (total volume of 150 μL)via a lateral tail vein using a 28-gauge insulin syringe. In the animalscontaining GFP-expressing tumor cells, Alexa-750 labeled peptide wasused due to its limited spectral overlap with GFP. Spectral fluorescenceimages were obtained using the Maestro™ FLEX In-Vivo Imaging Systemusing the appropriate filters for GFP (tumor; Ex445-490 nm, Em 515longpass filter; acquisition settings 500-720 in 10 nm steps), Texas Red(peptide; Ex 575-605 nm, Em 645; acquisition settings 630-850 in 10 nmsteps) or Alexa-750 (peptide; Ex 671-705 nm, Em 750 longpass filter;acquisition settings 730-950 in 10 nm steps). Acquisition settings were53 milliseconds for GFP and 1000 milliseconds for either Texas Red orAlexa-750 labeled peptide. Prior to peptide injection, background imageswere acquired through the skin to provide autofluorescence spectra.Following peptide injection, fluorescence images were acquired at 5 to15 minute intervals over the course of 2 to 3 hours. The multispectralfluorescence images were background subtracted and unmixed, usingMaestro™ software (Cambridge Research & Instrumentation, Inc. (CRi),Woburn, Mass.), to spectrally separate the autofluorescence animalsignal from the peptide signals. Regions of interest (ROI) were selectedover the tumor or non-tumor skin. Pixel values for the peptide signal,in photons measured at the surface of the animal, were determined withinthese ROI. Higher pixel values corresponded to presence of tumor. Pixelvalues in the tumor ROI were normalized to the non-tumor ROI and peptideconcentration, and then plotted. Each PTPμ peptide was tested on aminimum of three animals containing Gli36Δ5 and three animals containingLN-229 flank tumors. Statistical analyses were performed using MicrosoftExcel and an unpaired student t test.

Orthotopic Xenograft Tumors

Gli36Δ5-GFP cells were harvested for intracranial implantation bytrypsinization and concentrated to 1×10⁵ cells per μL of PBS. For braintumor implants, NIH athymic nude female mice were anesthetized byintraperitoneal injection of 50 mg/kg ketamine/xylazine and fitted intoa stereotaxic rodent frame (David Kopf Instruments, Tujunga, Calif.). Asmall incision was made just lateral to midline to expose the bregmasuture. A small (0.7 mm) burr hole was drilled at AP=+1, ML=−2.5 frombregma. Glioblastoma cells were slowly deposited at a rate of 1μl/minute in the right striatum at a depth of −3 mm from dura with a 10μL syringe (26G needle; Hamilton Company; Reno, Nev.). The needle wasslowly withdrawn and the incision was closed with sutures.

In Vivo Imaging of Intracranial Tumors

Nude mice with Gli36Δ5-GFP intracranial tumors were imaged at 9-12 daysafter GBM cell implant. Fluorophore-conjugated PTPμ peptides wereinjected via tail vein as described above. Following a 25 minuteincubation for clearance of unbound PTPμ peptide, the animals weresacrificed, the brains were removed and coronal sections made at 1 mmintervals. Individual brain sections containing tumor were placed on ablack slide and examined using the Maestro™ FLEX In-Vivo Imaging Systemas described above. Untreated brains containing Gli36Δ5-GFP intracranialtumors were used to provide autofluorescence spectra. ROI were selectedover the tumor region in each brain slice. Pixel values for the peptidesignal, in photons measured from the slice, were determined within theseROI. The multispectral fluorescence images were background subtractedand analyzed using the Maestro™ software as described above. Statisticalanalyses were performed using Microsoft Excel and an unpaired student ttest.

Results

An extracellular fragment of PTPμ is detected in human glioblastomas. Weshow a 55 kDa N-terminal extracellular fragment of PTPμ is retained inthe GBM samples, as shown by immunoblotting with an antibody against theextracellular segment of PTPμ (FIG. 1A). Based on size, and antibodyrecognition using antibodies against the MAM domain of PTPμ, thisfragment likely contains the MAM, Ig and the first two FNIII repeats(FIG. 2A). The 55 kDa extracellular fragment of PTPμ is retained in thecenter and edge of the resected GBM tumor but not substantially innormal cortical brain tissue from the same patient (FIG. 1A). The humanedge samples contain very few tumor cells proportionally to normalcells, suggesting that the PTPμ extracellular fragment is concentratedat the tumor edge and may be exploitable as a biomarker of the tumormargin.

Peptide Design and Optimization

The homophilic binding of PTPμ has been shown to require the domainsthat are present in the 55 kDa cleaved fragment (Brady-Kalnay S M, TonksN K (1994) Identification of the homophilic binding site of the receptorprotein tyrosine phosphatase PTPμ. J Biol Chem 269:28472-28477); ZondagG, Koningstein G, Jiang Y P, Sap J, Moolenaar W H, Gebbink M (1995)Homophilic interactions mediated by receptor tyrosine phosphatases μ andκ. J Biol Chem 270:14247-14250; Cismasiu V B, Denes S A, Reilander H,Michel H, Szedlacsek S E (2004) The MAM (meprin/A5-protein/PTPmu) domainis a homophilic binding site promoting the lateral dimerization ofreceptor-like protein-tyrosine phosphatase mu. J Biol Chem279:26922-26931.; Del Vecchio R L, Tonks N K (2005) The conservedimmunoglobulin domain controls the subcellular localization of thehomophilic adhesion receptor protein-tyrosine phosphatase mu. J BiolChem 280:1603-1612; Aricescu A R, Hon W C, Siebold C, Lu W, van derMerwe P A, Jones E Y (2006) Molecular analysis of receptor proteintyrosine phosphatase mu-mediated cell adhesion. Embo J 25:701-712;Aricescu A R, Siebold C, Choudhuri K, Chang V T, Lu W, Davis S J, vander Merwe P A, Jones E Y (2007) Structure of a tyrosine phosphataseadhesive interaction reveals a spacer-clamp mechanism. Science317:1217-1220; Aricescu A R, Siebold C, Jones E Y (2008) Receptorprotein tyrosine phosphatase micro: measuring where to stick. BiochemSoc Trans 36:167-172. Based upon crystallographic data indicating whichresidues are surface exposed (Aricescu A R, Hon W C, Siebold C, Lu W,van der Merwe P A, Jones E Y (2006) Molecular analysis of receptorprotein tyrosine phosphatase mu-mediated cell adhesion. Embo J25:701-712.; Aricescu A R, Siebold C, Choudhuri K, Chang V T, Lu W,Davis S J, van der Merwe P A, Jones E Y (2007) Structure of a tyrosinephosphatase adhesive interaction reveals a spacer-clamp mechanism.Science 317:1217-1220), and our binding studies (Brady-Kalnay S M, TonksN K (1994) Identification of the homophilic binding site of the receptorprotein tyrosine phosphatase PTPμ. J Biol Chem 269:28472-28477), wedesigned peptide probes to bind homophilically and recognize theadhesive domains contained within the cleaved fragment of theextracellular segment of PTPμ (FIGS. 2A, B). Four peptides derived fromthe MAM (SBK1 (SEQ ID NO: 4) and SBK2 (SEQ ID NO: 5) peptides) or Ig(SBK3 (SEQ ID NO: 6) and SBK4 (SEQ ID NO: 7) domains of PTPμ weregenerated (FIGS. 2A, B). As a control, a scrambled version of the SBK2peptide was also synthesized. The peptides were fluorescently labeledwith Texas Red-X succinimidyl ester dye on the N-terminus. Effectivepeptide concentrations for tissue labeling were determined by dilutionhistochemical analyses using human GBM tissue sections and indicatedthat a range of 3-40 μM was effective.

Detection of Human Glioblastomas with the PTPμ Peptide Probes

To examine the localization of the 55 kDa PTPμ fragment within GBMtumors, the Texas Red (TR) conjugated peptides were used as probes tolabel sections of GBM or noncancerous human brain obtained from epilepsypatients. The PTPμ-TR peptides, SBK2, SBK3 and SBK4 each recognized GBMtissue to varying degrees, although the best labeling occurred withpeptides SBK2 and SBK4 (FIGS. 3, 4). The SBK1 peptide did notsubstantially label either the noncancerous human brain or GBM (FIG. 3).The SBK2, SBK3 and SBK4 peptides typically labeled the parenchyma abovebackground with particularly bright labeling of the cell bodies.Endothelial cells lining blood vessels were also brightly labeled withinthe tumor (FIG. 4). Endothelial cells express high levels of PTPμ(Campman M, Yoshizumi M, Seidah N G, Lee M E, Bianchi C, Haber E (1996)Increased proteolytic processing of protein tyrosine phosphatase μ inconfluent vascular endothelial cells: the role of PC5, a member of thesubtilisin family. Biochemistry 35:3797-3802; Bianchi C, Sellke F W,Neel B G (1999) Receptor-Type Protein-Tyrosine Phosphatase mu IsExpressed in Specific Vascular Endothelial Beds in vivo. Experimentalcell research 248:329; Koop E A, Lopes S M, Feiken E, Bluyssen H A, vander Valk M, Voest E E, Mummery C L, Moolenaar W H, Gebbink M F (2003)Receptor protein tyrosine phosphatase mu expression as a marker forendothelial cell heterogeneity; analysis of RPTPmu gene expression usingLacZ knock-in mice. Int J Dev Biol 47:345-354; Koop E A, Gebbink M F,Sweeney T E, Mathy M J, Heijnen H F, Spaan J A, Voest E E, VanBavel E,Peters S L (2005) Impaired flow-induced dilation in mesentericresistance arteries from receptor protein tyrosinephosphatase-mu-deficient mice. Am J Physiol Heart Circ Physiol288:H1218-1223; Sui X F, Kiser T D, Hyun S W, Angelini D J, Del VecchioR L, Young B A, Hasday J D, Romer L H, Passaniti A, Tonks N K, GoldblumS E (2005) Receptor protein tyrosine phosphatase mu regulates theparacellular pathway in human lung microvascular endothelia. Am J Pathol166:1247-1258). PTPμ present on the surface of endothelial cells mayalso be proteolyzed in the tumor microenvironment. Two representativeGBM tumors are shown (Tumor 1 and Tumor 2). Similar results wereobserved in six different GBM samples (data not shown). In contrast,scrambled control peptide (Scrambled SBK2) did not label the GBM tissue(FIGS. 3, 4). When PTPμ peptides were incubated with sections ofnoncancerous brain tissue obtained from epilepsy patients, no specificstructures were labeled in either white or gray matter regions (FIGS. 3,4). Similar results were observed in six different samples ofnoncancerous “normal brain” (data not shown). PTPμ protein is highlyexpressed in normal brain and plays a role in stabilization of cell-cellcontacts (Burden-Gulley S M, Brady-Kalnay S M (1999) PTPμ regulatesN-cadherin-dependent neurite outgrowth. J Cell Biol 144:1323-1336;Burgoyne A M, Palomo J M, Phillips-Mason P J, Burden-Gulley S M, Major DL, Zaremba A, Robinson S, Sloan A E, Vogelbaum M A, Miller R H,Brady-Kalnay S M (2009b) PTPmu suppresses glioblastoma cell migrationand dispersal. Neuro-Oncology, March 20 Epub ahead of print). When PTPμis involved in cell-cell adhesion, its binding sites may be fullyengaged and therefore unavailable for recognition by the PTPμ peptides.Alternatively, in GBM tissue, the cleaved 55 kDa extracellular domain ofPTPμ may undergo a conformational change that allows recognition by thePTPμ peptides. Together, these results show that these PTPμ peptides canbe used for detection of human glioblastoma tissue.

The PTPμ peptides specifically recognize PTPμ. To confirm that the PTPμpeptides were binding to the 55 kDa PTPμ extracellular fragment in GBM,we performed antibody-blocking experiments. GBM tissue sections werepre-incubated with BK2 monoclonal antibody raised against the MAM domainof PTPμ (Brady-Kalnay S M, Tonks N K (1994) Identification of thehomophilic binding site of the receptor protein tyrosine phosphatasePTPμ. J Biol Chem 269:28472-28477) prior to incubation with the SBK2 orSBK4 peptides, which were generated from either the MAM or Ig domains ofPTPμ, respectively. Preincubation with BK2 monoclonal antibody caused acomplete block of SBK2 and SBK4 peptide binding (FIG. 5). Although SBK4is derived from the Ig domain of PTPμ, steric hindrance by the large BK2antibody likely prevented the SBK4 peptide from reaching its bindingsite on the Ig domain, which is in close proximity to the MAM domain,the target of BK2 (J Biol Chem 269:28472-28477). The PTPμantibody-mediated block of peptide binding suggests that the peptidesare recognizing the extracellular fragment of PTPμ in human GBM tissues.

Peptide recognition of glioblastoma associated proteins as a diagnostictool. Specific recognition of GBM cells in vivo through peptide bindingwould provide a powerful diagnostic tool. We recently observed adramatic reduction in full length PTPμ protein in the highly migratoryLN-229 human GBM cell line due to proteolysis (Burgoyne A M, Palomo J M,Phillips-Mason P J, Burden-Gulley S M, Major D L, Zaremba A, Robinson S,Sloan A E, Vogelbaum M A, Miller R H, Brady-Kalnay S M (2009b) PTPmusuppresses glioblastoma cell migration and dispersal. Neuro-Oncology,March 20 Epub ahead of print; Burgoyne A M, Phillips-Mason P J,Burden-Gulley S M, Robinson S, Sloan A E, Miller R H, Brady-Kalnay S M(2009a) Proteolytic Cleavage of PTPmu Regulates Glioblastoma CellMigration. Cancer Research In press). For this manuscript, an animalmodel system of GBM was developed for in vivo labeling. Two humanglioblastoma cell lines, LN-229 and Gli36Δ5, were injected individuallyinto the flanks of nude mice for the production of xenograft tumors. At2-3 weeks post-injection, the tumors were excised and homogenized forbiochemical analysis. The 55 kDa fragment of PTPμ is present in bothLN-229 and Gli36Δ5 cells when grown as flank tumors (FIG. 1B).

To observe localization of the 55 kDa fragment in the xenograft tumors,sections of fixed tumors were incubated with either SBK2 or SBK4peptides. The PTPμ peptides labeled both the Gli36Δ5 (FIG. 10) andLN-229 (FIG. 11) tumors, in a pattern that closely overlaid the cells ofthe tumors, as evidenced by GFP fluorescence, but also was observed insome intercellular spaces. This result is not unexpected since theextracellular fragment of PTPμ is likely shed by the tumor cells. Smallclusters of labeled cells were clearly visible in the tumormicroenvironment (FIG. 6), thus the peptides are useful for detection ofdispersing cells. The recognition of the 55 kDa fragment by PTPμpeptides suggested that this animal model was a viable system forfurther study in vivo.

Flank tumors provide a useful model for molecular imaging studies due toaccessibility and ease of imaging using fluorescence detection methods.Nude mice with Gli36Δ5 or LN-229 flank xenografts were imaged throughthe skin with the Maestro™ FLEX In-Vivo Imaging System using theappropriate filters for GFP, Texas Red or Alexa-750. Background imagesof the flank region containing the tumor were acquired, PTPμ peptideswere administered via tail vein injection, and the animals were imagedat regular intervals over the course of 2 to 3 hours. Fluorescence wasobserved throughout the animals within one minute after injection, butmaximal tumor labeling occurred within 10 to 20 minutes after injection.Of interest, the tumor microenvironment was also labeled, showing thatthe PTPμ fragment remains associated with the cells at the tumor edge.Unbound circulating peptide was cleared from the animals quickly,resulting in a clear demarcation of tumor over the normal tissuebackground (FIGS. 7A, 8A). In most cases, the tumor remained labeledabove background for at least 3 hours. The average signal in the tumorwas normalized to the average signal in non-tumor skin and plotted(FIGS. 7B and 8B). SBK2 peptide rapidly bound to the Gli36Δ5-GFP tumorand remained bound at a level greater than in the surrounding skin overthe course of the three-hour experiment (FIG. 7). Peak levels ofGli36Δ5-GFP tumor labeling were achieved by 10-20 minutes. Of note,similar results were obtained when the tumor was composed of LN-229-GFPhuman GBM cell line (FIG. 8), suggesting that the peptide may be usefulfor labeling glioblastomas in general. The SBK4 peptide labeled bothGli36Δ5 and LN-229 flank tumors to a similar extent as SBK2 but withdifferent off-rate kinetics (FIGS. 7B, 8B). The levels of Gli36Δ5 andLN-229 tumor labeling with SBK2 or SBK4 peptides were significantlydifferent from the scrambled control peptide, as analyzed with anunpaired student t-test. Two other peptides, SBK1 and SBK3, bound theGBM tumors poorly in vivo, with no significant difference from thescrambled control peptide.

The flank tumor labeling results provided proof of principle that thePTPμ peptides were capable of labeling GBM tumors in vivo. However, itwas important to determine whether these peptides could cross the bloodbrain barrier to reach physiological targets. Athymic nude miceimplanted with Gli36Δ5-GFP intracranial tumors were used for PTPμpeptide labeling experiments. SBK2-AL peptide was injected via tail veinand circulated in the mouse for 25 minutes to allow binding to theglioma brain tumor and initial clearance from non-tumor tissue. TheMaestro™ FLEX In-Vivo Imaging System cannot image through the skull.Therefore, the animals were sacrificed and the treated brain wasremoved, sectioned into 1 mm coronal slices and analyzed using theMaestro™ System as described above using filters appropriate for GFP andAlexa-750 fluorescence (FIG. 9). The location of the tumor in each sliceis indicated by GFP fluorescence (FIG. 9C). SBK2-AL peptide crossed theblood brain bather and remained bound to the glioma tumor (FIG. 9B).Note the high concentration of peptide binding in the region includingand directly adjacent to the Gli36Δ5 tumor (overlay image, FIG. 9D).These results show that the 55 kDa PTPμ fragment secreted by the tumorcells remains highly concentrated in the tissues surrounding the gliomatumor cells, as indicated by the overlap in fluorescence. Furthermore,the SBK2-AL was more effective in detecting small clusters of tumorcells than the GFP signal (FIGS. 9B and 9D section on the far right).SBK2-AL did not bind non-tumor brain (FIG. 9F). Quantitation of brainslice labeling in slices containing Gli36Δ5-GFP cells showed the SBK2-ALpeptide labeled tumor cells at a significantly higher level than eitherscrambled SBK2-TR or PBS control injections (FIG. 9G). Together, thesestudies show that PTPμ peptide probes SBK2 and SBK4 are useful asreagents to specifically label human glioblastoma tumors non-invasivelyin vivo.

From the above description of the invention, those skilled in the artwill perceive improvements, changes and modifications. Suchimprovements, changes and modifications within the skill of the art areintended to be covered by the appended claims. All references,publications, and patents cited in the present application are hereinincorporated by reference in their entirety.

Having described the invention, the following is claimed:
 1. A method ofdetecting cancer cells in a subject comprising: administering to asubject a molecular probe that includes a targeting agent that binds toproteolytically cleaved extracellular fragments of receptor proteintyrosine phosphatase (RPTP) cell adhesion molecules expressed in acancer cell microenvironment in the subject; and detecting whethercancer cells are present in the subject by detecting binding of themolecular probe to the proteolytically cleaved extracellular fragment ofthe cell adhesion molecule, wherein binding of the molecular probe tothe proteolytically cleaved extracellular fragment is indicative ofcancer cells in the subject.
 2. The method of claim 1, furthercomprising detecting in the subject the molecular probe bound to theproteolytically cleaved extracellular fragment of the cell adhesionmolecule to determine cancer cell migration, dispersal, and/or invasionin the subject.
 3. The method of claim 1, the RPTP cell adhesionmolecule comprising PTPμ.
 4. The method of claim 1, the cancer cellcomprising a glioma.
 5. The method of claim 1, the targeting agentcomprising a peptide that specifically binds to SEQ ID NO:
 2. 6. Themethod of claim 5, the targeting agent homophilically binding to SEQ IDNO:
 2. 7. The method of claim 3, the targeting agent being a peptidehaving an amino acid sequence selected from the group consisting of SEQID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO:
 7. 8. The method ofclaim 1, the targeting agent comprising an antibody or fragment thereofthat binds to the proteolytically cleaved extracellular fragments ofRPTP cell adhesion molecules.
 9. The method of claim 8, the antibody orfragment thereof specifically binding to SEQ ID NO:
 2. 10. The method ofclaim 1, the molecular probe further comprising at least one detectablemoiety, the detectable moiety comprising at least one of a isotopiclabel, radiolabel, metal label, or fluorescent dye.
 11. The method ofclaim 1, the molecular probe administered to the subject defining atumor margin of the cancer cells in the subject.
 12. The method of claim1, the molecular probe being administered parenterally to the subject.13. The method of claim 1, further comprising administering a cancertherapeutic to the subject after detection of the cancer cells.
 14. Themethod of claim 1, wherein the molecular probe is administeredpre-operative a surgical resection of cancer in the subject.
 15. Themethod of claim 1, wherein the molecular probe is administeredpost-operative a surgical resection of cancer in the subject.
 16. Amethod of treating cancer in a subject comprising: administering to asubject a molecular probe that includes a targeting agent that binds toproteolytically cleaved extracellular fragments of receptor proteintyrosine phosphatase (RPTP) cell adhesion molecules expressed in acancer cell microenvironment in the subject; detecting whether cancercells are present in the subject by detecting binding of the molecularprobe to the proteolytically cleaved extracellular fragment of the celladhesion molecule, wherein binding of the molecular probe to theproteolytically cleaved extracellular fragment is indicative of thelocation and/or distribution of the cancer cells in the subject; andadministering a cancer therapeutic to the subject and/or surgicallyresecting the cancer cells after detection of the cancer cells.